Secretory Products in Petals of Centaurea cyanus L. Flowers: A Histochemistry, Ultrastructure, and Phytochemical Study of Volatile Compounds

(1) Background: Centaurea cyanus L. is a medicinal plant whose flowers are widely used in herbal medicine. The aim of the study was to localise flower tissues that are responsible for the production of secretory products in petals and to analyse the volatile compounds. The volatile compounds of the flowers of this species have not been investigated to date. (2) Methods: Light, fluorescence, scanning and transmission electron microscopy techniques were used in the study. Lipophilic compounds were localised in the tissues using histochemical assays. Volatile compounds were determined with the use of solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). (3) Results: The study showed production of secretion in the petal parenchyma, whose ultrastructure has features of a secretory tissue. The lipophilic secretion was localised in the cells and intercellular spaces of the parenchyma and in the walls and surface of epidermal cells, where it accumulated after release through cuticle microchannels. Sesquiterpenes were found to constitute the main group of volatile compounds, with the highest content of β-caryophyllene (26.17%) and α-humulene (9.77%). (4) Conclusions: Given the presence of some volatile components that are often found in resins (caryophyllene, delta-cadinene) and the abundant secretion residues on the epidermal surface, we suppose that the C. cyanus secretion released by the flowers is a resinaceous mixture (oleoresin), which is frequently found in plants, as shown by literature data. This secretion may play an important role in the therapeutic effects of C. cyanus flowers.

Introduction
Plants emit different volatile organic compounds from their aerial and underground parts [1]. Most often they are produced by leaves and flowers of aromatic plants [2]. In flowers, volatile compounds are primarily synthesized in epidermal cells of petals, from which they are directly released into the atmosphere [1]. Volatile organic compounds serve various functions in flowers: attraction of pollinators or defence against herbivores and pathogens [3]. Secretory products of flowers are natural multi-component mixtures of volatile and non-volatile products. Terpenes, terpenoids, and molecules with an aromatic ring are present at high concentrations [4][5][6]. Volatile and semi-volatile compounds that are part of essential oils can constitute 85-99% of the entire oil fraction. Among them, there are hydrocarbon and derived mono-and sesquiterpenes, aliphatic and olefinic C 6 -C 12  The adaxial and abaxial epidermis of the ray florets was composed of elongated cells, with the central part of the surface exhibiting characteristic folding of the crested cuticle visible in the scanning electron microscope ( Figure 1C). The epidermis cells in the lobes of the ray florets were shorter, and the cuticle folds on their surface formed a crested pattern ( Figure 1D). In turn, the surface of the epidermis cell walls in the lower part of the ray florets was characterised by the presence of massive cuticle bands but no crests ( Figure 1E,F).
The scanning electron microscopy (SEM) revealed secretion deposits in some areas of the petal epidermis surface (Figure 1 E,F). No trichomes or papillae were found in the ray floret epidermis.

Secretory Activity of the Epidermis and Parenchyma
The cross sections of fused petals observed using the fluorescence microscope exhibited light blue autofluorescence of the epidermis cell cuticle. In some areas, secretion-containing vesicles in the distended cuticle emitted light blue fluorescence (Figure 2A). The blue, fluorescent secretion was also visible on the surface of the epidermis cells of fresh petals, where it formed linear clusters along the cuticle crests ( Figure 2B).
Lipid droplets emitting light green secondary fluorescence after the auramine treatment were visible on the surface of the petals, especially near the vascular bundles ( Figure 2C). The Sudan III treatment confirmed the lipid nature of the orange-stained droplets secreted by the epidermis cells ( Figure 2D). Fluorescence was also emitted by the secretion present in the intercellular spaces in the parenchyma and visible on the cross sections in permanent slides viewed under the fluorescence microscope after the auramine treatment ( Figure 2E-H). Fluorescence of secretion residues on the surface of epidermis cells was observed as well ( Figure 2I).
Starch grains are often observed in the cells of secretory tissues. We demonstrated their presence in parenchyma cells located around vascular bundles after the PAS reaction ( Figure 2J-L).

Ultrastructure of Secretory Cells
Both the epidermis and the parenchyma in the C. cyanus ray flowers served the secretory function. The outer wall of the epidermis cells had a considerable thickness (3.1 µm) and was covered by a relatively thin layer of a folded cuticle (0.36 µm) forming crests in the apical part ( Figure 3A-C). The central part of the cells was occupied by a large vacuole, and the dense cytoplasm was located parietally ( Figure 3A-D). The cytoplasm contained numerous ribosomes, mitochondria, smooth endoplasmic reticulum profiles, plastids, and numerous vesicles ( Figure 3B,D). The outer wall had a multi-layer structure ( Figure 3D-F,H), while the cuticle was formed by a homogeneous layer with microchannels ( Figure 3D-G). In some areas of the cell wall, there were dark, flocculent structures, which were likely part of the released secretion ( Figure 3E). This osmophilic substance accumulated mainly in the subcuticular part of the crests ( Figure 3E-G), and was then likely released through the cuticle microchannels and formed a layer with varying thickness on the surface ( Figure 3H).
The secretory parenchyma cells had irregular shapes ( Figure 4A,B). Large intercellular spaces filled with a dark heterogeneous substance were visible between the cells ( Figure 4B-E). As in the case of the epidermis cells, the cytoplasm formed a thin parietal layer, and the central part of the cells was occupied by large vacuoles (Figure 4A-C). The dense cytoplasm contained numerous mitochondria ( Figure 4D), ribosomes ( Figure 4F-I), and plastids with dark stroma and well-developed internal tubules ( Figure 4F,G). Additionally, there were dictyosomes, numerous different-sized vesicles ( Figure 4H), and endoplasmic reticulum profiles mainly located close to the plasmalemma ( Figure 4I). Multivesicular bodies, likely originating from the cytoplasm, were visible in the vacuoles ( Figure 4B,G). permanent slides viewed under the fluorescence microscope after the auramine t ( Figure 2E-H). Fluorescence of secretion residues on the surface of epidermis observed as well ( Figure 2I).
Starch grains are often observed in the cells of secretory tissues. We demo their presence in parenchyma cells located around vascular bundles after the PAS ( Figure 2J-L).  ( Figure 3D-G). In some areas of the cell wall, there were dark, flocculent st were likely part of the released secretion ( Figure 3E). This osmophilic sub lated mainly in the subcuticular part of the crests ( Figure 3E-G), and wa leased through the cuticle microchannels and formed a layer with varyi the surface ( Figure 3H).  plastids with dark stroma and well-developed internal tubules ( Figure 4F ally, there were dictyosomes, numerous different-sized vesicles ( Figure 4 plasmic reticulum profiles mainly located close to the plasmalemma (F tivesicular bodies, likely originating from the cytoplasm, were visible in the ure 4 B,G).  , parietal cytoplasm and large intercellular spaces (asterisks) with a dark substance; (B) a cell with walls with varying thickness containing multivesicular bodies in the vacuole and parietally located cytoplasm. The intercellular spaces (asterisks) are filled with a dark substance/secretion; (C) large intercellular spaces filled with a dark substance/secretion along the entire length of some walls (asterisks); (D) fragments of cells with numerous different-sized vesicles (arrows) and mitochondria (m) in the cytoplasm and secretion in the intercellular spaces (asterisks); (E) fragments of cells with dense cytoplasm and a heterogeneous substance/secretion in the intercellular space (asterisk); (F) a plastid (p) with dark stroma, tubular internal structure, and small vesicles in the periplasmic space (arrow); (G) multivesicular body (mb) directed towards the vacuole, plastid (p) with dark stroma and small vesicles in the periplasmic space (arrows); (H) dictyosome (ds) in the dense cytoplasm; (I) smooth endoplasmic reticulum (er) profile and numerous small vesicles located close to the plasmalemma (arrows) and fibrillar material in the intercellular space (asterisk). Scale bars: 5 µm (A), 2 µm (B-D), 1 µm (E), 0.5 µm (F-I).

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts wer tected in the C. cyanus ray florets. The absence of secretory ducts in floret petals wa ported in several other species from the family Asteraceae, e.g., Matricaria chamomilla Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Cent species had large biseriate glandular hairs on the corolla surface. A similar type o chomes was detected on the corolla in other species of this family: Helichrysum sto [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc flore C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc fl were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanu florets released fragrant secretory products. Floral tissues secreting such products osmophores, produce trichomes and papillae in the epidermis, or the epidermis is posed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.

Secretory Activity of Corolla Tissues
No such secretory structures as papillae, glandular trichomes, and ducts were detected in the C. cyanus ray florets. The absence of secretory ducts in floret petals was reported in several other species from the family Asteraceae, e.g., Matricaria chamomilla [16], Santolina ligustica [15], Centaurea rupestris, and C. fritschii [14]. However, these Centaurea species had large biseriate glandular hairs on the corolla surface. A similar type of trichomes was detected on the corolla in other species of this family: Helichrysum stoechas [32], Chamomilla recutita [16,33], and Inula helenium [34]. Centrally located disc florets in C. cyanus inflorescences were found to have only papillae [20]. Papillae on disc florets were also observed in other genera of this family: Flourensia [35] and Petasites [36].
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.
The present study demonstrated that the epidermis cells of petals in the C. cyanus ray florets released fragrant secretory products. Floral tissues secreting such products, i.e., osmophores, produce trichomes and papillae in the epidermis, or the epidermis is composed of cells with a simple cubic form [37]. The epidermis of C. cyanus ray florets is an osmophore similar to the latter type.
The outer wall of the epidermis cells in the C. cyanus ray florets (tribe Cardueae) exhibited a crested cuticular pattern, which was also observed in an earlier study [20]. The present comprehensive study demonstrated the presence of the pattern only in the apical and central parts of these florets. In turn, longitudinal cuticular striation of the epidermis surface was observed in the lower part of the ray florets. The crested cuticular pattern has also been observed in Asteraceae species belonging to other tribes, e.g., Cichorieae and Mutisieae [38,39].
As shown in the cross sections, the outer cell walls of the petal epidermis cells were very thick (3.1 µm), and there were microchannels in the cuticle layer. The secretion was released through the wall in the area of the cuticular crests, as shown by fluorescence microscopy and transmission electron microscopy (TEM). The secretion transported via vesicles from the distal part of the cell to the periplasmic space reached the epidermis surface by crossing the cell wall and microchannels in the cuticle. A similar transport of aromatic secretions via microchannels into the environment was observed in the osmophores of Orbea variegata [40] and Passiflora superosa [41,42]. However, cuticular diffusion related to the lipophilic nature of the cutin is recognized as the most common way of releasing fragrance by plants [37,43,44]. Some plants (Acianthera) have been described to release secretory products (e.g., essential oil) through stomata [45].
In the epidermis cells of the fresh ray florets, different-sized lipid drops were shown by both the light microscope (Sudan III) and the fluorescence microscope. In turn, on the surface of the epidermis of these florets, residues of a copious secretion covering the outer cell wall were shown in the fixed material by SEM and TEM. The presence of the secretion was likely associated with its high viscosity and the fact that not all of its components were volatilised. Therefore, it can be assumed that C. cyanus ray florets secrete resinaceous material.
The cells of the subepidermal parenchyma of the ray florets had many features of scent-producing cells: high cytoplasmic density, cytoplasm rich in ribosomes, smooth endoplasmic reticulum, plastids with dark stroma, numerous lipid droplets, dictyosomes, many mitochondria, and vesicles, which were often localised near the plasmalemma. These traits are recognised as characteristic for osmophores by many authors [41,44,46]. The smooth endoplasmic reticulum profiles in the C. cyanus flower cells were located near the plasmalemma in the peripheral part of the cytoplasm. As reported by Skubatz et al. [47], endoplasmic reticulum (ER) structures in Sauromatum can associate with the cell membrane during secretion of volatile sesquiterpenes. The presence of large intercellular spaces in the parenchyma containing heterogeneous lipid material, likely the secretion, should also be regarded as one of the traits of glandular tissues.
The presence of a lipid-containing substance in the intercellular spaces of the parenchyma in the ray florets indicates the apoplast route of secretion in these flowers. As reported by other authors, lipid droplets may be transported in small vesicles to periplasmic and intercellular spaces [45]. In many plants, the exchange of substances between secretory cells occurs via vesicles and/or via plasmodesmata (symplast route) [37]. In the case of the C. cyanus ray florets, there were no plasmodesmata in the parenchyma cell walls.
It was shown previously that lipids, which are components of such a secretory product as essential oil, are formed in plastids [37,48]. Other authors have reported that secretion/essential oil droplets often originate from plastids, periplastidal reticulum, and smooth reticulum but sometimes from dictyosomes or other organelles [11]. The connection between the metabolic pathways in the cells of various organs and the site of terpenes synthesis indicates that plastids are mainly responsible for the formation of volatile mono-(C 10 ) and diterpenes (C 20 ) [49]. In turn, the cytosol, ER, and peroxisome pathway leads to the formation of precursors of volatile sesquiterpenes (C 15 ) [3,50,51].

Volatile Compounds of Petals
Most literature data present the composition of essential oil in different Centaurea species obtained from aerial plant parts [26,[28][29][30]. Our research is the first to show the composition of the most volatile compounds collected from Centaurea cyanus fresh corolla of flowers.
β-caryophyllene has been reported by various authors as an important essential oil component from Centaurea species growing in Asia. The content of this compound is species-dependent and varies from 8.1% (in C. pseudoscabiosa subsp. pseudoscabiosa) to 33.9% (C. depressa) [31,[52][53][54][55]. A high concentration of β-caryophyllene in essential oil obtained from Centaurea ragusina flowers was also presented by Politeo [26].
It is remarkable that only one monoterpene derivative (bornyl acetate) was detected in the investigated volatile compounds. This compound does not occur in Centaurea species growing in the Middle East (Egypt or Iran) [26,28,29,31,[52][53][54][55]. However, it can be found in plants originating from Tatarstan (Centaurea scabiosa. flowers) [57] or the central Balkans (Centaurea orientalis and Centaurea atropopurpurea) [58]. Thus, the presence of bornyl acetate in Centaurea plants seems to be dependent on the latitude. This compound has also been detected in the resin of two species of the Taxodium genus [59].
Detailed data on the biological activity of Centaurea cyanus and other flower extracts from Centaurea species have been presented lately by Sharonova et al. [57]. Antimicrobial and antioxidant effects have been shown in the case of Centaurea cyanus L. from Kosovo (antimicrobial activity) [60] and Romania (antioxidant properties) [61].
In the present study, we have shown that sesquiterpenes are the main group of components in the volatile compounds from C. cyanus flowers. Literature data show that, together with sesquiterpene lactones and monoterpenes, sesquiterpenes are characteristic constituents of essential oil and resin in the family Asteraceae. Hence, these compounds may be important for the chemotaxonomy of this family [62,63]. The results of the present study are, to a certain extent, similar to the data on other taxa from this family.
In our studies, the secretion in C. cyanus flowers forms large clusters on the petal epidermis surface visible in scanning electron microscopy. These remnants of the secretory product may be sesquiterpenes, which are semi-volatile compounds that are characterised by low volatility and easily adhere to plants' surfaces, as reported in the literature [1].
Additionally, it is known that the odoriferous secretion released by organs of various plant species frequently contains essential oil associated with resins or gums [9,64]. This seems to be the case of C. cyanus flowers, as their secretion exhibits the characteristics of a resinaceous substance. Fresh ray florets (30) were randomly chosen for the morphometric analyses from 20 inflorescences.

Light Microscopy
The material for microscopic examinations consisted of 10 florets chosen randomly from 10 inflorescences. Handmade cross sections of the lower, middle, and upper parts of the fresh florets were prepared for preliminary analyses using razor blades. Observations of semi-permanent slides prepared in glycerin with water (1:1) were carried out with the use of a Nikon Eclipse 400 light microscope. An alcoholic Sudan III solution (POCH, Gliwice, Poland) was used for detection of total lipids in the fresh material [66].
Semi-thin sections were prepared from the same areas of the ray florets. Fragments of corollas (5 × 5 mm) were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.2; 0.1 M) for 12 h at 4 • C. In the next step, the samples were washed three times in phosphate buffer, dehydrated in an ethanol series, and embedded in LR white resin (LR white acrylic resin, medium grade, Sigma-Aldrich, Saint-Louis, MO, USA). The semi-thin sections (0.7-0.9 µm) were cut longitudinally and transversally using a Reichert Ultracut S ultramicrotome (C. Reichert Optische Werke AG, Vienne, Austria) and a glass knife. For general histology, the sections were stained with a 1% (w/v) aqueous methylene blue-azure II solution [67]. The presence of insoluble polysaccharides was detected using Periodic Acid-Schiff's (PAS) staining [67] after blocking free aldehyde groups. The sections were examined using an Olympus CX 23 light microscope (Olympus, Tokyo, Japan) equipped with an Olympus EP50 digital camera (Olympus) and EPview software.

Fluorescence Microscopy
The autofluorescence of ray floret tissues and sections was observed using a Nikon Eclipse 90i fluorescence microscope (Nikon, Tokyo, Japan) with UV-2 B filter equipped with a digital camera (Nikon Fi1) and NIS-Elements Br 2 software. Secondary lipid fluorescence was investigated after treatment of the semi-thin sections with auramine O [68]. The staining reaction was examined using a fluorescein isothiocyanate filter. Control sections were used in each case.

Transmission Electron Microscopy (TEM)
Sections (4 × 4 mm) from different ray florets (n = 5) were fixed as described above and treated for 1.5 h with a 1% osmium tetraoxide solution at 0 • C. The samples were washed with distilled water, and graded ethanol series were used for dehydration. Next, the plant material was saturated in 1:3, 1:1, and 3:1 mixtures of LR White resin (LR white acrylic resin, medium grade, Sigma-Aldrich, Saint-Louis, MO, USA) and acetone for 3 h each. After embedding in LR White resin, the samples were cut into ultra-thin sections (from 60 to 90 mm) with the use of the Reichert Ultracut Microtome. The ultrastructure of the cells was observed with the use of a JEM 1400 (JEOLL Ltd., Tokyo, Japan) transmission electron microscope at an accelerating voltage of 120 kV equipped with an 11 Megapixel TEM Camera MORADA G2 (EMSIS GmbH, Münster, Germany).

Scanning Electron Microscopy
For observations of the epidermis surface, small pieces of ray florets (about 3 × 3 mm) were fixed in a 2.5% or 4% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.0) at room temperature for 2 h and then washed in phosphate buffer four times at 20-min intervals. The fixed plant material was dehydrated in graded ethanol series and immersed in absolute ethanol (POCH, Gliwice, Poland) three times for 30 min. Next, the floret samples were critical-point dried in liquid CO 2 using a K 850 Critical Point Dryer and sputter-coated with gold (20 mm thickness) using K 550X (Emitech, Ashford, UK). The observations were carried out using a Tescan Vega II LMU scanning electron microscope (Tescan, Brno, Czech Republic) at an accelerating voltage of 30 kV [69].

SPME Extraction of Floral Volatile Components
A sample (2 g) of freshly collected flowers of C. cyanus was introduced into the SPME (Solid Phase Microextraction) glass thimble. The SPME sorption fibre (made from polydimethylsiloxane, PDMS, diameter 30 µm, Sigma-Aldrich, Saint-Louis, MO, USA) was slotted into the shredded raw material for 30 min. The extraction was performed at room temperature. Next, the system was placed in the injector of a gas chromatograph (description presented in part GC-MS determination), where the thermodesorption (at 250 • C for 3 min) took place. The volatile compounds were then separated on a chromatographic column. A dispenser was used to perform the determinations (split 1:50; T = 250 • C). The temperature program for SPME was as follows: 35 • C for 3 min and then 8 • C/min to 250 • C (final isotherm 1 min).

GC-MS determination
The composition of the Centaurea cyanus L. volatile compounds was determined with the GC-MS technique (ITS-40 apparatus (GC/ITMS system), Finnigan MAT, Temecula, CA, USA) with an RT-5 capillary column (Resteck, Bellefonte, PA, USA) 20 m long, 0.18 mm in diameter, and stationary phase film thickness of 0.25 µm. The MS analysis was performed using electron ionization (70 V) with a 35-500 m/z.
Qualitative analysis was carried out by comparison of the MS spectra with the NIST spectral library (National Institute of Standards and Technology, Gaithersburg, MD, USA) [70] and the spectral library of the UMCS Analytical Laboratory (Lublin, Poland). Identity of the volatile compounds was confirmed by their retention indices, which were determined in relation to a homologous series of n-alkanes (C 7 -C 18 ). The quantitative composition of volatile compounds was determined assuming that the sum of individual compounds is 100%.

Conclusions
This study is the first report on the constituents of volatile compounds in fresh petals of C. cyanus flowers (ray florets). The main group of aroma components was constituted by sesquiterpenes, with the highest content of β-caryophyllene and α-humulene.
The microscopic analyses facilitated localisation of the sites of secretion production, i.e., parenchyma cells. The secretion was transferred via vesicles to the cells of this tissue and intercellular spaces and then to epidermal cells. Next, it penetrated the surface of these cells through cuticle microchannels, most often in the crested pattern zone. The present study showed the function of the crested cuticle present in the flowers of some Asteraceae, which was involved in the release of secretory products in C. cyanus.
The physical properties and chemical composition of the secretion suggest that cornflower flowers secrete a mixture containing a resinaceous substance.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.


Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 μg/mouse) of depolymerase intraperitoneal injection protected 90%–100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.

Introduction
Klebsiella pneumoniae is ubiquitous as it is found in animals and ecological environment. It causes septicemia, pneumonia, soft tissue infections, urinary tract infections, surgical site infections and catheterrelated infections in human, particularly among immunocompromised peoples (Podschun and Ullmann, 1998). Besides, it also causes clinical mastitis in cow (Medrano-Galarza et al., 2012). The increasing carbapenem-resistant K. pneumoniae (CRKP) led to the loss of effectiveness of antibiotics against these infections, and made it become one of the most troublesome Gram-negative bacteria in the world (Gu et al., 2018;Pitout et al., 2015;Xu et al., 2017). Thus, the Centers for Disease Control of the USA designated K. pneumoniae as an urgent threat to public health (CDC, 2019;Ventola, 2015).
K. pneumoniae is typically coated with a thick capsule polysaccharide (CPS) layer, which takes part in the evasion of the host defense system (Li et al., 2014). CPS also contributes to biofilm formation (Li et al., 2014;Wu et al., 2011). Capsule removal increases susceptibility to gentamicin in vivo (Bansal et al., 2014). From 1926 to 1977, a total of 77 capsular types were identified by serological reactivity tests (Ørskov and Fife-Asbury, 1977). From 2008, some new capsular types, KN1-KN5, have been defined by the CPS gene clusters in K. pneumoniae Pan et al., 2008Pan et al., , 2013Pan et al., , 2015Pan et al., , 2017. Capsule locus type (KL) KL47 and KL64 CRKPs are increasingly prevalent in China (Li et al., 2020a;Yang et al., 2020). Additionally, a part of ST11-K47 and K64 CRKP isolates are hypervirulent, which poses great challenges to antimicrobial therapy (Gu et al., 2018;Huang et al., 2018;Yang et al., 2020). Under this background, it is an urgent need to seek alternatives to conventional antibiotics to prevent and control severe CRKP infections.
Depolymerase Dep42 derived from phage SH-KP152226 specific for K47 capsule was able to significantly inhibit biofilm formation or degrade formed biofilms. Capsule depolymerases were also demonstrated to be potential anti-virulence agents that act by defeating resistance mechanism of K. pneumoniae against the innate immunity (Lin et al., 2014;Majkowska-Skrobek et al., 2018). Depolymerase also increases the lifespan of Galleria mellonella larvae (Majkowska-Skrobek et al., 2018) and survival rates of mice infected with K. pneumonia Lin et al., 2014;Pan et al., 2015;Wang et al., 2019). As more depolymerases were discovered, it will help to use synthetic biology to produce and design phage-derived proteins instead of whole phage particles showing a broader antibacterial spectrum, better tissue penetration, lower immunogenicity and low probability of bacterial resistance in the future (Drulis-Kawa et al., 2015).
In this study, we reported and characterized a Klebsiella phage P560 infecting KL47 K. pneumoniae, and identified a phage-derived capsule depolymerase specific for KL47 types CPS. Furthermore, we also investigated the potential application of depolymerase in the inhibition of biofilms as well as the prevention and control of CRKP infections.

Bacteria and growth conditions
A total of 25 different KL types K. pneumoniae, including 9 CRKP (Li et al., 2020a) and 7 non-CRKP isolates from human and 9 K. pneumoniae from cows, used in this study were listed in Table 1. A total of 24 KL47 K. pneumoniae used in this study were listed in Supplementary Table S1. Multilocus sequence typing was used to investigate the sequence types (STs) (Diancourt et al., 2005). KLs were determined by wzi sequencing (Brisse et al., 2013). All isolates were routinely cultured in Luria-Bertani (LB) broth at 37 C on orbital shaker with 180 rpm. Tryptic soy broth medium (TSB, Merck, Germany) were used to culture bacterial biofilms. Phosphate Buffer Saline (PBS, 0.1 mol/L Na 2 HPO 4 , 0.15 mol/L NaCl 2 , pH 7.2) was used for dilution and wash of K. pneumoniae cultures.

Morphology of the phage
Phage P560 was isolated from sewage water in a hospital in Suzhou, China using CRKP Kp42 as host strain (Li et al., 2020a). The counting and propagation of the phage was performed by double-layer agar plate method (Adams, 1959). SM buffer [50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 10 mmol/L MgSO 4 and 0.01% gelatin] was used for the dilution of phages. Phage suspensions were purified and stored as previous methods described (Li et al., 2021). Examination of bacteriophage morphology was performed by transmission electron microscopy (Li et al., 2020b).

Genome sequence analysis
Phage suspensions were concentrated and purified for genomic DNA extraction as previously described (Sambrook and Russell, 2006). Phage genome was sequenced using the Illumina HiSeq system (Illumina, USA). Sequencing reads were de novo assembled using Spades 3.11.1 (Bankevich et al., 2012). Annotation of the phage genome was conducted by the RAST server (Aziz et al., 2008), followed by manual curation. The whole-genome sequence of phage P560 was deposited in the GenBank database. The closely related phages to P560 were searched using online BLASTn against the NCBI database (https://blast.ncbi.nlm.nih.gov/Bla st.cgi). Genome alignment of these closely related phages was illustrated using EasyFig to analyze their similarities and differences. Further analysis of the product (putative tail fiber protein, TFP) of ORF43 gene from phage P560 was carried out using the Phyre2 server (Kelley et al., 2015).

Cloning, expression and purification of putative capsule depolymerase
To generate N-terminal (His) 6 -tagged capsule depolymerase proteins, the depolymerase ORF43 gene (including stop codons) were inserted into the pET-28a expression vector (Novagen, Madison, WI, USA) via the Nde I and Xho I restriction sites. Primers used to amplify the capsule depolymerase gene are: forward, 5 0 -GGAATTCCA-TATGTTAAACAATCTGAATCAG-3 0 ; and reverse, 5 0 -CCGCTCGAGT-TATGGACCAATGACCACACC-3 0 . PCR amplification program was 95 C  for 3 min, followed by 30 cycles of 95 C for 15 s, 50 C for 15 s and 72 C for 2 min. Then the PCR products were ligated into the pET-28a expression vector after restriction enzyme digestion. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) cells, and expression of recombinant proteins was induced by the addition of 0.3 mmol/L IPTG and the mixture was incubated at 16 C overnight. The resulting His-tagged protein, P560dep, was purified using nickel beads (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instructions.
The purified protein was concentrated by centrifugation using a 30-kDa MW cut-off (MWCO) membrane (Thermo Scientific, USA) and stored at À80 C. The concentration of the purified proteins was determined by the BCA Protein Assay Kit (Thermo scientific, USA). The purified proteins were analyzed in 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels stained with Coomassie blue (Thermo Scientific, USA). A molecular weight (MW) standard (10-180 kDa) (Thermo Scientific, USA) was used for SDS-PAGE.

Activity spectrum of phage P560 and depolymerase
Spot testing often causes an overestimate of the host range (Mirzaei and Nilsson, 2015). Therefore, we used double-layer agar plate test to determine the host range of phage P560. A total of 25 clinical isolates of different KL types mentioned above were used to examine the host range of phage P560.
Log-phase bacterial cultures of K. pneumoniae were poured onto an LB soft agar overlay plate (LB with 0.5% agar) to form lawns. Purified P560dep protein was assessed for its polysaccharide-degrading activity with the concentrations from 2 mg/mL to 0.2 ng/mL on Kp42 CRKP strains (10 μL) using spot assay. PBS buffer was used as a negative control. The sensitivity of all 25 different KL types K. pneumoniae to P560dep (20 μg/mL) was determined by the single-spot assay.

Depolymerization activity against capsular polysaccharide
The bacterial capsular polysaccharide was extracted according to a previous method (Domenico et al., 1989). Depolymerization activity of P560dep against KL47 capsular polysaccharides was determined using our previous method (Li et al., 2021). All experiments were performed in triplicate. To investigate depolymerization activity against capsules, transmission electron microscopy was performed according to a previous study (Li et al., , 2021.

Animal inoculation and capsule depolymerase treatment
Five-week-old specific-pathogen-free female BALB/c mice were procured from the Laboratory Animal Center of Yangzhou University (Yangzhou, China). All efforts were made to minimize the suffering of animals. Mice were divided into four groups (10 mice in each group), and each group was kept in two clear polypropylene cages with free access to a standard antibiotic-free food and water.
In the treatment group, mice were infected with 2.5 Â 10 7 CFU Kp42.
After 1 h of the bacterial challenge, mice were given 50 μg P560dep by intraperitoneal injection. Meanwhile, two control groups were administered with the same volume of K. pneumoniae strain Kp42 plus PBS buffer, and only PBS buffer, respectively. All groups were monitored for 7 days. To assess the effects of a single P560dep injection, heart, liver, spleen, lungs, kidney and brain were collected, and fixed in 4% paraformaldehyde buffer. Samples were dehydrated with a dehydrator, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Further observation was carried out under a light microscope for histological analyses.

Statistical analysis
All statistical analyses in this study were carried out using the GraphPad Prism 5 software package (GraphPad, LaJolla, CA, USA). Oneway ANOVA was used for biofilm assay and the bacterial survival assay in mice whole blood. For mice experiments, survival rates were analyzed with the log-rank test. Differences at P < 0.05 were considered significant.

Morphological characterization of phage P560
Phage, named as P560, can lyse KL47 CRKP strains. Phage P560 formed big clear and round plaques with a diameter of approximately 0.3 cm surrounded by 0.1 cm enlarged haloes, indicating that they may encode depolymerases with polysaccharide-degrading activity (Fig. 1A). Transmission electron microscopy confirmed that it belonged to the order Caudovirales. The diameter of capsid of phage P560 was estimated at 50 AE 0.5 nm (Fig. 1B).

Genome analysis of phage P560
Next generation sequencing analysis showed that phage P560 was a linear dsDNA molecule of 40,562 bp and a GC content of 53.1%, and harbored 47 putative coding sequences. No lysogeny or virulence associated genes were identified in the genome of phage P560, showing its lytic nature and the potential for therapeutic application. Two TFPs, ORF43 and ORF44, were identified in phage P560 based on the RAST predication.
Online BLASTn analysis of P560 genomic sequence revealed that it is mostly close to Klebsiella phage KP32i196 (Accession no.: NC_047971) isolated from wastewater in Novosibirsk Oblast, Russian Federation, with 84% query coverage and 93.4% nucleotide identity and Klebsiella phage SH-KP152226 (MK903728) isolated from sewage in Shanghai, China, with 90% query coverage and 92.7% nucleotide identity. The results of genome structure and sequence similarity all suggested that phage P560 belonged to the genus Przondovirus, family Autographiviridae, the order Caudovirales.
The TFP ORF43 gene of P560 had low homology with that of phage KP32i196, high sequence identity to that of Klebsiella phage SH-KP152226 ( Fig. 2A). Besides, it showed high sequence identity to that of Klebsiella phage vB_KpnP_IME205 and Klebsiella phage IME304. The enzyme activities of the TFPs mentioned above have not been examined. Phyre2 analysis revealed that ORF43 exhibited functional and structural similarity to galacturonase (96.4%; 11%), and lacked the N-terminal domain of Gp17 in T7 phage (Dunn and Studier, 1983) (Fig. 2B). The whole-genome sequence of phage P560 was deposited in the GenBank database under accession number MT966873.

Expression and activity spectrum test of P560dep
The recombinant depolymerase P560dep were successfully expressed in E. coli BL21 cells. The purified recombinant depolymerase P560dep migrated as a single band with an approximate MW of 70 kDa on a 10% SDS-PAGE gel (Fig. 3A). The purified depolymerases were diluted to 2 mg/mL using PBS buffer. Phage P560 only can form plaques on KL47type strains Kp42 by double-layer agar plate method. The recombinant depolymerase P560dep exhibited capsule-digesting activity against KL47-type K. pneumoniae, indicating that activity spectrum of P560dep matched to the host range of phage P560 (Table 1). However, depolymerase P560dep only can degrade 19 out of 24 KL47 capsule of K. pneumoniae (Supplementary Table S1).

Depolymerization activity of P560dep
The agar plate showed that depolymerase P560dep was active against Kp42 strain of KL47 capsule. The translucent halo zones of P560dep were detected at the concentrations as low as 0.2 μg/mL (Fig. 3B). The results of punch tests revealed that the holes treated with P560dep presented   2. Comparative genome analysis, and prediction of TFP structure and function. A. Schematic was produced using Easyfig_2.2.3_win software based on the whole genome nucleotide sequences of phage P560 compared to closely related phages KP32i196 (NC_047971) and SH-KP152226 (MK903728). TFP genes were showed as red arrows. Vertical blocks between sequences indicate regions of shared similarity shaded (blue and red for matches in the same direction or green and yellow for inverted matches; identity: 70%-100%). B. Prediction and analysis of TFP structure and function. The product of ORF43 gene (TFP) was analyzed by the Phyre2 server. degradation-rings, due to CPS degradation. In contrast, when the holes were treated with PBS buffer (as a control), no degradation-rings appeared (Fig. 3C).
Transmission electron microscopy showed significant differences on the morphology characteristics of P560dep treated group and PBS control group. The surface of Kp42 treated with P560dep was smooth (Fig. 4A), suggesting that "capsule-stripped" bacteria were generated. The surface of Kp42 treated with PBS was rough (Fig. 4B).

Antibiofilm activity of the recombinant depolymerase P560dep
To further test the impact of depolymerase P560dep on biofilm formation, we investigated the inhibition effect of P560dep with different concentrations on exponential-phase bacteria Kp57. The results of crystal violet staining assay showed that the OD 595 values of five different P560dep-treated groups (4 μg, 0.4 μg, 0.04 μg, 4 ng and 0.4 ng) decreased significantly in comparison to that of untreated group (Fig. 5, P < 0.001).  M. Li et al. Virologica Sinica 37 (2022) 538-546 These results suggested that when P560dep ! 0.4 ng, it can significantly inhibit biofilm formation.

Depolymerase prevention and treatment in mouse infections
To verify the protection effect of depolymerase P560dep, three groups of BALB/c mice (n ¼ 10) infected with bacteria were then treated with P560dep before and after infection. The results showed that 9 of 10 mice infected with 2.5 Â 10 7 CFU of KL47 CRKP Kp42 strain died within one days without depolymerase P560dep treatment. In contrast, mice treated with 50 μg P560dep before infected with Kp42 all survived (P < 0.001); nine of the 10 infected mice treated with 50 μg enzyme at 2 h post-infection survived (P < 0.001, Fig. 6A). Intraperitoneal administration of 2.5 Â 10 7 CFU of CRKP Kp42 in mice led to bacteremia within 8 h (Fig. 6B). In contrast, intraperitoneal administration of a single dose of (50 μg/mouse) depolymerase P560dep 8 h post infection resulted in complete elimination of bacteria in the blood (Fig. 6B). H&E staining showed infiltration of neutrophils in lung and vacuolation in liver of Kp42 infected mice treated with PBS, however, no obvious pathological changes in mice treated with P560dep (Fig. 6C). Besides, no obvious pathological changes were observed in heart, spleen, kidney and brain tissues in either group ( Supplementary Fig. S1). These results demonstrated that depolymerase P560dep can prevent and treat CRKP infections in mice.

Discussion
The world-wide prevalence of carbapenem-resistant K. pneumoniae poses a big threat to the public health. In China, most of CRKPs belonged to the clonal complex CC11, especially K64/KL64 and K47/KL47 types of ST11 (Chen et al., 2018a;Li et al., 2020a;Yu et al., 2019;Zhang et al., 2017). In this study, we isolated phage P560, and identified depolymerase P560dep targeting KL47 K. pneumoniae. Previous studies also reported phage SH-KP152226 , IME205  and SRD 2021 (Hao et al., 2021) specific for CRKP with capsular K47 serotype. Most Klebsiella phages belonged to Autographiviridae, Drexlerviridae and Myoviridae family. Przondovirus genus in Autographiviridae family and Webervirus in Drexlerviridae family were the main parts of all reported Klebsiella phages (Walker et al., 2021). Phage P560 belonging to the genus Przondovirus showed high genome sequence similarity to phage KP32i196 and SH-KP152226, and ORF43 gene of phage P560 also showed high sequence similarity to that of phage SH-KP152226. However, the enzyme activity of ORF43 from SH-KP152226 has not been analyzed yet.
Conservative phage therapy brings with drawbacks, including rapid emergence of phage-resistant bacteria and the potential for horizontal gene transfer of virulence and drug resistance genes, etc (Labrie et al., 2010). Hence, some phage-derived enzymes are highly valued. In Gram-positive bacteria, phage-derived endolysins can be applied exogenously, presenting a clear advantage over the whole-phage application (Kaspar et al., 2018). It also proved to have protective efficacy in different animal models (Entenza et al., 2005;Gilmer et al., 2013;Nelson et al., 2001) and human clinical trials (Totte et al., 2017). However, in Gram-negative bacteria, the outer membrane prevents endolysins from accessing and degrading the underneath peptidoglycan layer, thereby protecting them from endolysin attack (Lai et al., 2020). Therefore, the application of phage-derived depolymerases as one of the promising alternative approaches for Gram-negative bacteria infection, gained increasing attention to explore their potential as antimicrobial agents. In this study, it was demonstrated that phage P560 can infect KL47-type K. pneumoniae, while capsule depolymerase P560dep had capsule depolymerization activity against KL47-types capsule of K. pneumoniae. Except the depolymerases identified for 24 capsular types of K. pneumoniae mentioned above, future studies will be carried out to identify novel depolymerases for other 58 capsular types of K. pneumoniae. It is confirmed that the capsule depolymerases are host specificity determinants of these phages (Pan et al., 2019). P560dep treatment also led to the generation of "capsule-stripped" bacteria, which also demonstrated the polysaccharide-degrading activity of depolymerase P560dep targeting KL47 capsule. However, depolymerase P560dep cannot degrade all KL47 capsule of K. pneumoniae, that is, 5 out of 24 KL47-type strains (Kp5, Kp6, Kp9, Kp18 and Kp28) were insensitive to phage P560 and its depolymerase (Supplementary Table S1), which may be due to the subtle differences in capsule composition that existed within KL47 serotype. This phenomenon was similar to a recent report that two depolymerases (Dpo42 and Dpo43) from Klebsiella phage IME205 targeted different subsets of K47 capsule .
Bacterial biofilms are communities of bacteria and surrounded selfproduced matrix composed of polysaccharides, proteins, lipids and nucleic acids (Flemming and Wingender, 2010). Biofilm plays an important role in the evasion of the host immune system and antimicrobial agents, and is a leading pathogenic factor in the medical devices and clinical settings (Clegg and Murphy, 2016). Phage-derived polysaccharide depolymerases were valuable for inhibiting and/or eradicating Klebsiella biofilms , E. coli O157 biofilms (Park and Park, 2021) and Shiga toxin-producing Escherichia coli biofilms . In this study, P560dep can inhibit biofilms formation of KL47 CRKP Kp42 stains. Additionally, it have been demonstrated that various depolymerases reduced colonization of the surfaces by interfering with the formation of Proteus mirabilis biofilms (Rice et al., 2021), Acinetobacter baumannii biofilms (Shahed-Al-Mahmud et al., 2021) and Klebsiella biofilms (Chai et al., 2014;Chen et al., 2020). In fact, depolymerase-resistant mutants emerged during the treatment of K. pneumoniae biofilm, which resulted in beneficial consequences, i.e., the sensitization to the innate immune response (Kaszowska et al., 2021).
M. Li et al. Virologica Sinica 37 (2022) 538-546 the treatment of the recombinant capsule depolymerase P560dep also effectively reduced bacteremia and deaths in mice infected with KL47 K. pneumoniae. A single dose (i.p., 50 μg/mouse) of P560dep exhibited both preventative (100%) and therapeutic efficacy (90%) against the bacterial infection. Although capsule depolymerase alone is not sufficient to destroy the bacterial outer membrane and kill the bacteria, its unique ability to degrade bacterial capsular material can be applied to decrease pathogen virulence and expose the underlying bacterium to the immune attack such as complement-mediated killing (Majkowska-Skrobek et al., 2018). The complete eradication of bacteria in blood and the therapeutic efficacy on reducing deaths in animals infected by K. pneumoniae may be because depolymerases striped capsules and exposed the underlying bacteria to innate immune attack. Considering the potential of phage-derived depolymerase P560dep as an anti-virulence compound for antimicrobial therapy, its three dimensional structure and catalytic mechanism need to be determined in future study.

Conclusions
Phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae with the enlarged haloes around plaques. We demonstrated that a capsule depolymerase from Klebsiella bacteriophage P560, had depolymerization activity against KL47-types capsule of K. pneumoniae, and its lysis spectrum matched to the host range of phage P560. Recombinant depolymerase can significantly inhibit biofilm formation, prevent and treat CRKP infection in mouse models. Depolymerase as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.

Data availability
The annotated whole-genome sequences of phage P560 were deposited in the GenBank database under accession number: MT966873.

Ethics statement
All animal studies were approved by the Experimental Animal Welfare and Ethics Committee of Nanjing Agricultural University (Approval ID: PZB2020002), and were performed according to Animal Welfare Agency Guidelines.

Author contributions
Min Li: conceptualization, data curation, formal analysis, validation, investigation, writing original draft, writing-review and editing. Hui Wang: data curation. Long Chen: resources. Genglin Guo: formal analysis. Female BALB/c mice were infected with 2.5 Â 10 7 CFU bacteria and treated with 50 μg P560dep 1 h before (prevention group, P < 0.0001) and 2 h after infection (treatment group, P ¼ 0.0004) (10 mice in each group). Control group was administered with the same volume of K. pneumoniae strain Kp42 plus PBS buffer. Statistical analysis was performed using the Kaplan-Meier method. The x axis represents days after inoculation, and the y axis represents the survival rates of mice. B.
Bacterial load (CFU) in the blood of mice 8 h post infection. Mice were infected via intraperitoneal route with 2.5 Â 10 7 CFU CRKP Kp42 and treated with 50 μg P560dep. Error bars represent SEM from independent values. C. Depolymerase P560dep alleviated pathological changes of lung and liver in mice infected with CRKP Kp42. Representative H&E-stained lung and liver were shown. Scale bar ¼ 50 μm.

Conflict of interest
All authors declare that they have no conflict of interest.

Acknowledgement
This study was supported by grants from the National Natural Science


s2fieldsofstudy: Biology Medicine

text:
Innate immune activation as cofactor in pemphigus disease manifestation

Molecular mechanisms underlying auto-antibody-induced acantholysis in pemphigus vulgaris are subject of current research to date. To decipher the discrepancy between ubiquitous antibody binding to the epidermal desmosomes, but discontinuous disease manifestation, we were able to identify Ultraviolet A (UVA) as a cofactor for acantholysis. UVA induces interleukin (IL)-1 secretion in keratinocytes, mirroring innate immune system activation. In an in vitro keratinocyte dissociation assay increased fragmentation was observed when UVA was added to anti-Desmoglein 3 Immunoglobulins (anti-Dsg3 IgG). These results were confirmed in skin explants where UVA enhanced anti-Dsg3-mediated loss of epidermal adhesion. The UVA-mediated effect was blocked in vitro by the pan-caspase-inhibitor zVAD-fmk. Thus, we introduce UVA as a caspase-dependent exogenous cofactor for acantholysis which suggests that local innate immune responses largely contribute to overt clinical blister formation upon autoantibody binding to epidermal cells in pemphigus vulgaris.

Introduction
Pemphigus vulgaris (PV) is a chronic autoimmune blistering disease characterized by the production of autoantibodies against Desmogleins (Dsg) 3 and Dsg1 causing dissociation of keratinocytes in the cell complex (acantholysis) and subsequent blister formation of the mucous membranes and the skin (1-3). At present, the complex mechanisms causing acantholysis are not fully understood but are known to require signaling mechanisms besides direct inhibition of desmoglein binding (4). PV still poses a therapeutic challenge due to severe co-morbidities of current treatment options, a refractory course (5,6) and a high mortality when untreated (7). Although antibodies bind ubiquitously to the epidermis, acantholytic blisters only occur at distinct areas of the skin and the mucous membranes (8). Exogenous cofactors activating the innate immune system might be a key element in explaining the discrepancy between acantholysis and a lack of blister formation. Accordingly, in blister fluid from PV patients, increased levels of innate cytokines were detected which suggests a pathogenic role of innate immune activation (9-11). There are hints that IL-1a can increase complement activation which is observed in a majority of PV patients and might play an important role in acantholysis (12). Additionally, the innate immune system can modulate T cell-mediated disorders such as psoriasis (13) and presumably also PV (14).
IL-1 is an inflammasome-related proinflammatory cytokine involved in auto-immune and auto-inflammatory processes [reviewed by (15)]. It connects innate and adaptive immune responses by orchestrating lymphocyte differentiation (16).
Innate contributors in the pathogenesis of blistering diseases belonging to the pemphigoid-group have been early voiced due to the rich lesional inflammatory infiltrate of neutrophils, eosinophils and lymphocytes (17). Bullous pemphigoid (BP) is proposed to be a T-cell dependent, mainly Th2-like autoimmune disease with production of pathogenic IgG antibodies against the BP180 ectodomain of hemidesmosomes (18). This was based on the discovery of increased Th2-related (19, 20) cytokines in BP patients. Increased levels of complement factors (21-23), innate cytokines IL-1a and IL-1b (24,25), Th-type cytokines IL-3, IL-4, IL-6, IL-10, and Granulocyte-macrophage colony-stimulating factor were detected in the sera and/or blister fluids from BP-patients (26).
The interaction of the innate and adaptive immune system and the release of cytokines are poorly understood in PV. This study aims to dismantle the local impact of cytokines on celldissociation apart from their role as key mediators in cellmediated processes of autoantibody production in PV. Altered T-cell subsets are known to fuel the immuno-pathogenesis and inflammation in the skin [reviewed by (27,28)]. Related to this, extensive changes in the cytokine network of pemphigus patients were described [reviewed by (29)]. Previous studies provided initial evidence for a local role of innate cytokines: IL-1-deficient mice as well as tumor necrosis factor-alpha receptor-deficient mice showed decreased susceptibility to PV-related antibodies (10). In addition, IL-1 upregulated tissue-type plasminogen activator secretion in the spontaneously immortalized keratinocyte cell line HaCaT (30), which might also be involved in PV-related acantholysis (31).
To improve treatment options and to obtain a better understanding of PV, we investigated whether IL-1-inducing stimuli, such as UVA irradiation led to an activation of the innate immune system as a cofactor for blister formation in PV. Here, we were able for the first time to identify UVA as a caspasedependent exogenous cofactor in the pathogenesis of PV.

Augmentation of acantholysis via inflammatory caspases UVA induces protein secretion of innate cytokines
To determine the effect of UVA irradiation as a potential cofactor in PV, the spontaneously immortalized keratinocyte cell line HaCaT was irradiated with UVA at 5 J/cm² (the titrated intensity that resulted in the best cytokine response, while cell viability was not affected, Supplementary Figure 1). Afterwards, cells were treated with IgG purified from PV sera (PV-IgG) which contained high levels of anti-Dsg3 IgG. Subsequently, gene expression of proinflammatory cytokines IL1A, IL1B, IL6 and IL8 was determined via real-time PCR (rtPCR) and the secretion of respective cytokines was measured by Enzymelinked Immunosorbent Assay (ELISA).
After treatment with UVA, HaCaT cells showed a two-fold increase in mRNA expression for IL1A, IL1B only but a robust induction of IL6 and IL8 compared to untreated cells ( Figure 1A). The treatment with PV-IgG did not lead to any notable changes in the expression of the indicated cytokines which is in line with previous findings (32). Furthermore, UVA irradiation induced the secretion of IL-1a, IL-1b, IL-6 and IL-8. Stimulation with PV-IgG alone did not result in a significant increase in cytokine release ( Figure 1A).
Combined stimulation with UVA followed by PV-IgG treatment showed no significant differences in cytokine gene expression and in protein secretion except for IL-1b compared to the stimulation with UVA alone.

UVA enhances PV-IgG-induced acantholysis caspase-dependent in vitro
Next, we evaluated the pathogenicity of anti-Dsg3 antibody and UVA as a pro-inflammatory cofactor in vitro. Therefore, we applied a well-established dispase-based keratinocyte dissociation assay on HaCaT cells. Principle of this method is to determine the cohesive strength of a keratinocyte monolayer due to stimulation, application of mechanical stress and quantification of resulting fragments (33). Again, the secretion of IL-1 was determined by ELISA. To block inflammasomemediated caspase activation, the pan-caspase-inhibitor zVadfmk was added prior to treatment with UVA and antibody.
As expected, treatment with the specific anti-Dsg3 IgG AK23 led to fragmentation of the cell monolayer as a surrogate parameter of acantholysis. The previous irradiation with UVA (subpathogenic level) significantly increased the number of fragments ( Figure 1B). This effect was reversed by treating cells with the pan-caspase inhibitor zVAD-fmk prior to UVA-irradiation and treatment with anti-Dsg3 antibody, verifying the involvement of caspase activation. In the dissociation assay, additional UVA treatment significantly enhanced the secretion of IL-1a and IL-1b compared to the untreated control condition. This effect was less pronounced in costimulation with AK23 but was reversed by pretreatment with pan-caspase inhibitor zVAD-fmk.

Innate immune system activation enhances PV-IgG mediated blister formation ex vivo
To mimic a more physiological condition, skin explants taken from the safety margins of tumor operations were treated with UVA (15 J/cm²) and subsequently anti-Dsg3 antibody AK23 for 12 hours. As previously shown, the treatment with PV-IgG led to suprabasal acantholysis (34). (A) HaCaT cells were irradiated with 5 J/cm 2 UVA. Directly afterwards IgG purified from patient sera was added. After an incubation period of four hours, the expression of IL1A, IL1B, IL6 and IL8 was determined via rtPCR. Protein secretion was measured by ELISA. (B) After irradiation of HaCaT (5 J/cm 2 UVA) the monoclonal anti-Dsg3 antibody AK23 was added for four hours. To block the effect caspase-activation zVad-fmk was added one hour prior to irradiation. A dispase-based keratinocyte dissociation assay was performed by applying shear stress on the epidermal monolayers. Cell fragments were stained with MTT and counted, IL-1 secretion was measured via ELISA. (C) Human skin explants were irradiated with 15 J/cm² UVA and incubated ex vivo for 48 hours with or without the monoclonal anti-Dsg3 antibody AK23 in increasing doses. Afterwards explants were HE stained. Error bars represent the SEM. *p < 0.05. The data evaluation was carried out via a One-way analysis of variance. **** p<0.0001; ** p<0.01; * p<0.05 Here, we titrated the concentration of the anti-Dsg3-antibody AK23 to a subpathogenic concentration, which alone did not induce acantholysis. After irradiation with a non-toxic dose of UVA (35), the threshold to induce acantholysis by anti-Dsg3 IgG was lowered and suprabasal blister formation was observed histologically ( Figure 1C). UVA irradiation alone did not result in morphological alterations.

Discussion and perspectives
PV is considered a paradigm of an IgG autoantibodytriggered autoimmune disease of the skin which is mainly regulated by cellular components of the adaptive immune system (36). Antigen-specificity and immunological memory make it unique and enable to distinguish from the innate immune system (37). Thus, recent findings reveal that innate immune activation contributes to various autoimmune diseases (38) as e.g. in psoriasis through modulation of T cells (13). The primary immune system as first line of defense against invading danger fulfills immuno-protective functions on the one hand, but once being over-activated is the origin of inflammatory skin diseases. It reacts through the detection of external signals, cytokines, and chemokines, released by inflammatory cells. There are several extra-und intracellular pattern recognition receptors whose activation lead to cytokine release, with Nodlike receptors (NLR) being one of them (39). NLRs upon activation form inflammasomes, intracellular multi-protein complexes which result in caspase-activation. Caspases are proteases involved in cell death and inflammatory responses. Activation of caspase-1 through inflammasome formation leads to pyroptosis (a pro-inflammatory form of cell death) and cleavage of inactive pro-IL-1b to biologically active IL-1b, leading to its secretion (37) (Figure 2). In addition, caspase-1 independent mechanisms of IL-1 release were identified, too (40,41).
To understand the pathogenesis of autoimmune blistering dermatoses and specifically PV, the role of the immune system in the loss of self-tolerance and initiation of autoantibody production (38), and a local influence of the antibodies on the epidermis, finally resulting in acantholysis, have to be clearly distinguished. Aim of the study was to focus on factors needed for blister induction upon antibody binding to the epidermis.
It is known that incubation with anti-Dsg3 IgG can induce acantholysis both in vitro and in vivo (42,43). Our results show that the threshold of acantholysis can be lowered by an exogenous cofactor, which in the case of UVA acts via activation of the innate immune system. Previous hints for a possible pathophysiological involvement were obtained by detecting elevated levels of innate cytokines from the blister fluid and sera of PV patients (9, 10, 44). We propose local activation of the innate immune system as a possible cofactor in autoantibody-induced acantholysis in the pathogenesis of PV. Specifically, we identified UVA to be a caspase-dependent pathogenic factor.
UVA irradiation of keratinocytes leads to innate immune activation and the secretion of IL-1 and IL-6, important cytokines of the innate immune system (45).
Innate cytokines are known to negatively impact autoimmune diseases (e.g. lupus erythematosus) (46) and were further described as a trigger in the manifestation of pemphigus foliaceus (47). Increased secretion of proinflammatory cytokines upon UV treatment in PV patients has been described previously (48). In our case, the treatment with IgG purified from PV Model of cofactor influenced acantholysis in PV. The local activation of the innate immune system through activation of TLRs and inflammasomes or via activation of caspases is known in keratinocytes. patient sera led to notable changes in expression of the abovementioned cytokines. Furthermore, UVA irradiation induced the secretion of IL-1a, IL-1b, IL-6 and IL-8 ( Figure 1A).
Interestingly, keratinocyte monolayers treated with UVA dissociate in significantly more fragments upon mechanical stress in the presence of anti-Dsg3 IgG compared to monolayers treated with anti-Dsg3 IgG only. We are proposing UVA irradiation with subsequent activation of the innate immune system as a cofactor inducing acantholysis after antibody binding in vitro. These results were confirmed by an ex vivo human skin explant model. Here, UVA also decreased the antibody threshold for anti-Dsg3-mediated acantholysis as the combination of PV antibody and UVA led to acantholysis at a lower antibody concentration compared to untreated skin explants.
Although, UVA cannot entirely explain the occurrence of cutaneous symptoms at the predilection sites (8) as oral mucosa is not exposed to the sun, we detected an obvious impact on acantholysis under experimental conditions. Other exogenous factors related to the activation of the innate immune system may also be pathogenically relevant and should be investigated in further experiments.
Muller et al. argued that the induction of cytokine production by keratinocytes does not contribute to the pathogenesis of PV but are rather induced via a secondary activation of the innate immune system by a disturbed epidermal barrier (49). However, as the level of innate cytokines in the serum as well as in the blister fluid of PV patients obviously reflects the dynamics of the disease process and we observed that UV radiation induced cytokine secretion and enhanced autoantibody-mediated acantholysis, these data indicate that cytokines contribute to diseases activation. Additionally, in clinical remission, the levels of innate cytokines decrease, while the amount of endogenous IL-1 receptor antagonist increases (44). Furthermore, stimulation of PBMC in patients with endemic pemphigus foliaceus resulted in higher IL-1b secretion compared to healthy donors (50). These clinical observations have been supported by experimental data showing that IL-1 receptor (IL-1R) knockout mice were protected from blister formation after the injection of PV serum. While injection of pathogenic IgG autoantibodies against Dsgs into newborn mice induced intraepidermal loss of adhesion, this phenotype is absent in mice deficient for IL-1R type 1, the receptor for IL-1a and IL-1b (10).
The functional network of different caspases and cytokines is closely interwoven, and its complexity is yet poorly understood. Whether IL-1 is the cause or the consequence of increased acantholysis remains cryptic and needs to be addressed in future studies. Based on our findings, we propose an additive effect through activation of the innate immune system on acantholysis. Importantly, under experimental conditions, we characterized the UVA-mediated effect being caspase-dependent because the pan-caspase-inhibitor zVAD-fmk was effective to block the effect of UVA on acantholysis. This is interesting because in previous studies in the same cell line zVAD-fmk did not reduce loss of cell adhesion when PV-IgG was applied in the absence of UVA (51). Thus, the additional effect of UVA may explain why in several mouse models a contribution of caspase signaling to acantholysis was observed (52,53). Furthermore, desmosomes themselves could also play a role in local modulation of the innate immune system (49).

Concluding remarks
In future studies, the role of the innate immune system as a cofactor in the pathogenesis of PV and the influence of cytokines on desmosomes should be investigated further. Besides the direct effects of innate cytokines, the impact of caspases needs to be elucidated. All in all, we are the first to introduce the local activation of the innate immune system via inflammatory caspases as a possible cofactor in the pathogenesis of autoantibody-induced acantholysis in PV, which might act as starting point for further studies on disease manifestation of autoantibody-mediated diseases.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethics statement
The studies involving human participants were reviewed and approved by the ethical approval: 547/2011BO2, University of Tuebingen, Germany. The patients/participants provided their written informed consent to participate in this study.
Author contributions RE, MS, and AY contributed to the conception and design of the study, supported by MH, JW, and JMB. RE, MS, and EW performed the experiments. RE, MS, and AY wrote the manuscript. All authors contributed to the manuscript revision, read, and approved the final version.

Funding
This work is funded by the grant from the DFG (Deutsche Forschungsgemeinschaft) to the Research Unit FOR 2497 PEGASUS (to AY and JB (both TP3), JW (TP4) and MH (CP and TP8)).